Cholinesterase is a term which refers to one of the two enzymes: Acetylcholinesterase (AChE), and Pseudocholinesterase (BChE or BuChE), also known as plasma cholinesterase, or butyrylcholinesterase. Both of these compounds catalyze the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation.
In 1968, Walo Leuzinger successfully purified and crystallized the enzyme from electric eels at Columbia University, NY. Several cholinesterases are commercially available typically from animal or marine sources such as horse, cow, electric eels and the like. Cholinesterase is relatively instable and is best kept at low temperatures. Some attempts to stabilise the enzyme by immobilizing it on a porous surface or platform as in U.S. Pat. No. 6,406,876 or dehydrating the enzyme so it can be safely stored and later rehydrated for use as in U.S. Pat. No. 4,324,858 have been attempted. In actual use it is, however difficult to obtain a sensitive, simple, portable, economic kit or method for rapid detection of cholinesterase inhibitors without resorting to cumbersome steps for the improvement of shelf life.
A cholinesterase inhibitor (or “anticholinesterase”) suppresses the action of the enzyme. Because of its essential function, chemicals that interfere with the action of cholinesterase are potent neurotoxins, causing excessive salivation and eye watering in low doses, followed by muscle spasms, respiratory failure, convulsions and ultimately death. Examples of cholinesterase inhibitors are some snake venoms, organophosphate pesticides, and the nerve gases sarin, Soman, Tabun, Cyclosarin VR and VX.
During the Sarin incident in the Tokyo subway in 1995, an incredible influx of over 600 casualties reported to one of the hospital's Emergency Department within the initial hours of the incident. In all, the hospital's Emergency Department received a total 640 patients, of which an overwhelming 99.2% did not require emergency treatment. 82.5% patients had only mild symptoms and hence could be classified under the category of mass psychogenic illness. Such a huge influx of patients into hospitals following any chemical nerve agent event is likely to be a common aftermath of future terrorist actions and is expected to severely reduce the efficiency of any Emergency Department. An efficient and rapid screening procedure would be required at the hospital triage stage to differentiate those requiring medical attention due to moderate and severe exposure; inclusive of dermal exposure, from amongst the worried-well or psychogenic masses.
As symptoms of chest tightness and headache arising from mass psychogenic reaction cases resemble clinical signs of mild to moderate inhalation exposure to nerve agents, there exists a genuine difficulty in distinguishing such “worried-well” cases from exposed casualties by clinical symptoms alone. The current diagnostic kit for monitoring an individual's exposure to nerve agents/organophosphorous pesticides is by monitoring for a 30% decrement in blood cholinesterase from baseline values. Such kits include EQM Testmate and automated laboratory cholinesterase assays from Walter Reed Army Institute of Research. However, these biomonitoring techniques require prior establishment of the individual's baseline acetylcholinesterase levels, which limits their application in sudden terrorist incidents involving nerve agents or organophosphorus (OP) inhibitors. Moreover, upon administration of pre-treatment medication, pyridostigmine by first responders, inter-individual variance in degree of blood cholinesterase inhibition (10-50% inhibition), complicates efforts to diagnose subsequent exposure to nerve agents using blood cholinesterase measurements. There is hence a paucity of definitive, rapid mass triage diagnostic devices to resolve similar mass casualty problem as encountered during the Tokyo Subway Sarin Attack.
Amongst non-exposed individuals, intra-individual variation in erythrocyte AChE activities could be as much as 13% while that between individuals could range from 75-150% of mean population baseline AChE level. These normal values are so wide that it is possible for patients to have substantial decline in erythrocyte AChE level and yet stay within the population normal range. During organophosphate exposures, appearance of clinical symptoms is also more dependent on the rate of decline in AChE level than on the absolute level of erythrocyte AChE observed. Hence, while most cases manifest clinical symptoms when erythrocyte AChE is inhibited by 70-80%, clinical manifestations have also been recorded at 40-50% erythrocyte AChE inhibition. On the other hand, symptoms may also appear following sudden acute exposure associated with a rapid drop of synaptic AChE even when erythrocyte AChE inhibition is less than 30%. Moreover, as erythrocyte AChE level is also known to be affected by nutritional or water deficiency and pregnancy, a threshold of 30% inhibition of erythrocyte AChE has been selected as a reference level for indicating possible organophosphate exposure. In the absence of clinical symptoms, erythrocyte acetylcholinesterase (AChE) inhibition less than 30% has little utility for confirming OP exposure nor could it rule out possible toxic sequela following exposure. In view of the inherent fluctuations in the level of this natural biomarker, monitoring of erythrocyte AChE level has serious limitations for diagnosis of low-level OP exposure, which may record AChE inhibition less than 30% and little or no clinical signs and symptoms of OP poisoning. Knowing each individual's pre-exposure AChE baseline level would assist in diagnosis of low level OP exposure but this is not feasible in terrorist attack scenarios. The eye's response, on the other hand, while being more responsive than AChE inhibition to OP, is not confirmatory of organophosphate exposure as miosis can also be produced by barbiturate overdose.
One of the most common classes of cholinesterase inhibitors are phosphorus-based compounds which bind to the active site of the enzyme. The structural requirements are a phosphorus atom bearing two lipophilic groups, a leaving group such as a halide or thiocyanate and a terminal oxygen.
Cholinesterase inhibitors are also used in anesthesia or in the treatment of myasthenia gravis, glaucoma and Alzheimer's disease. Also such compounds are used for killing insects in a range of products including sheep dip, organophosphate pesticides, and carbamate pesticides. In addition to acute poisoning as described above, a semi-acute poisoning characterized by strong mental disturbances can occur. Also, prolonged exposure can cause birth defects. The toxic nature of organophosphate, organosulfur and carbamate pesticides has led to several regulatory requirements to protect people who handle such pesticides. This includes monitoring occupational exposure to the pesticide by routinely measuring the blood cholinesterase levels of people handling the pesticides. A current portable diagnostic method available known as the Test-mate system, requires each individual's pre-exposure cholinesterase baseline level. This is not feasible in situations where there were previously no regulations in a particular country or area and an individual has been exposed for many years without having had their pre-exposure baseline cholinesterase level tested.
Generally if someone is suspected of having been exposed to a cholinesterase inhibitor, treatment protocols dictate that treatment should be instituted based on appearance of clinical symptoms and without waiting for test results of laboratory tests. As symptoms of chest tightness and headache arising from mass psychogenic reaction cases resemble clinical signs of mild to moderate inhalation exposure to nerve agents, there is a possibility of inappropriate antidote administration to non-intoxicated patients. The most common treatments include atropine and oximes. Oximes such as pralidoxime and Hagedorn oximes such as obidoxime and HI-6, themselves interfere with cholinesterase levels in the blood through their actions on cholinesterase inhibitors or through direct effects on cholinesterases. This can interfere with conventional measurement of cholinesterase levels in the blood as an indicator of exposure to a cholinesterase inhibitor and prevent retrospective diagnosis post-antidotes administration.
Upon intoxication with organophosphorus (OP) chemicals, these agents bind rapidly to cholinesterases in blood, and hence intact free agents in blood disappear soon after exposure, causing difficulty in detecting the free OP chemicals. However, retrospective analysis is made possible using reactivation method with fluoride ions, as demonstrated in regeneration of these protein-bound nerve agent sarin in blood samples of alleged sarin victims of Tokyo (1995) and Matsumoto (1994) terrorist attacks (Martine Polhuijs, et. al. New Method for Retrospective Detection of Exposure to Organophosphorus Anticholinesterases: Application to blood samples of alleged Sarin Victims of Japanese Terrorists. Toxicology and Applied Pharmacology 1997; 146: 156-161). This method focuses on detection of the cholinesterase inhibitor present in a sample rather than the conventional test for cholinesterase level of a sample. However, the method is laboratory bound as it required the use of solid-phase extraction or liquid-liquid extraction and analysis by sophisticated expensive equipment like GC-MS. The technique takes about 20 minute per sample and it would take more than 3 hours to complete 10 samples. It is not suitable for rapid field tests in a terrorist situation and would be a very expensive monitor for occupational exposure to pesticides.
The present invention seeks to provide a rapid method for detection of cholinesterase inhibitors as an indication of exposure to the cholinesterase inhibitor. It provides a kit for carrying out the methods.
General
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.
Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.
The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.